Project #1: Elimination of mossy cell NMDA receptors results in impaired emotional reactivity.[unreadable] Mossy cell NR1-KO mice and their control littermates were subjected to a battery of behavioral tasks including fear conditioning, a task that involves associating a particular context with an aversive stimulus. We found that the mutants show reduced freezing to a single foot-shock as compared to their control littermates. Further, they are unable to discriminate training and novel contexts 3 hours after fear conditioning. Interestingly, when the foot shock was intensified, these phenotypes disappeared, suggesting that impaired context discrimination is due to the deficit in immediate post-shock freezing. The mutants were also impaired in a contextual step-through avoidance task using a weak foot-shock. In addition, we found significantly decreased rearing activity in the mutants in the open field. However, there was no impairment in home cage activity, tactile and pain perception, motor coordination, or anxiety/despair-like behavior. Collectively, these results suggest that the behavioral deficits observed in the mutants is not due to anxiolytic hyperactivity or central analgesia, but perhaps due to decreased emotional reactivity to weak invasive stimuli. Cellular analysis of hilar mossy cell synapses is underway to explore the underlying mechanisms.[unreadable] [unreadable] Project #2: Elimination of mossy cells impairs contextual pattern separation but does not induce epileptogenesis.[unreadable] In mossy cell-DTR mice, intraperitoneal. injection of diphtheria toxin (DT) induces the ablation of DTR-expressing cells. We confirmed that, after DT injection for two consecutive days, the mutant mice exhibit significant neurodegeneration of mossy cells within a week and approximately 90% of mossy cells were selectively eliminated by 4 weeks after the treatment. We hypothesized that the selective removal of these cells may lead to an increased susceptibility to epilepsy and we conducted local field potential recordings from awake behaving mutant mice to explore this possibility. We found no obvious or sustained epilepsy-like discharges in the hippocampus. No mossy fiber sprouting was detected by Timm staining. Behaviorally, no apparent seizure-like behavior was detected. These results suggest that, in contrast to previous reports showing that lesions of the entire hilar region induce massive mossy fiber sprouting and epilepsy, selective in vivo elimination of mossy cells does not trigger behavioral epilepsy or mossy fiber sprouting. Our results rather suggest mild dentate hyperexcitability as an acute effect, implying that mossy cells play an inhibitory role in the dentate gyrus network in vivo. Electrophysiological recording from dentate granule cells is underway to evaluate granule cell hyperexcitability directly.[unreadable] How does the increased dentate granule cell excitability affect physiology and, ultimately, behavior? We evaluated the pattern separation capability in these mutants, a hallmark of dentate function, using a context discrimination task in the fear conditioning paradigm. When trained and then tested with a contextual discrimination task 5-7 days after the DT injection, the mutants were unable to discriminate the training from a novel context. Interestingly, the mutants were normal when tested 35 days after the DT injection, suggesting that the pattern separation deficit and its effect on memory formation and expression appear only when dentate excitability is increased. This result suggests that mossy cells play a role in dentate granule cell excitability, which is critical in executing the dentate gyrus functions.